Cancer is the second leading cause of death in the United States and over 1.2 million people are diagnosed with this disease annually. Cancer is significant, not only in lives lost, but also in the $107 billion cost to the United States economy in 2000 according to the National Institutes of Health. It is widely recognized among the cancer research community, that there is a need to develop new tools to characterize normal, precancerous, cancerous, and metastatic cells and tissues at a molecular level. These tools are needed to help expand our understanding of the biological basis of cancers. Molecular analysis of tissue changes in cancer improve the quality and effectiveness of cancer detection and diagnosis strategies. The knowledge gained through such molecular analyses helps identify new targets for therapeutic and preventative agents.
Diagnosis of cancer is the first critical step to cancer treatment. Included in the diagnosis is the type and grade of cancer and the stage of progression. This information drives treatment selection. When cancer is suspected, a patient will have the tumor removed or biopsied and sent for histopathology analyses. Conventional handling involves the tissue undergoing fixation, staining with dyes, mounting and then examination under a microscope for analysis. Typically, the time taken to prepare the specimen is of the order of one day. The pathologist will view the sample and classify the tissue as malignant or benign based on the shape, color and other cell and tissue characteristics. The result of this manual analysis depends on the choice of stain, the quality of the tissue processing and staining, and ultimately on the quality of education, experience and expertise of the specific pathologist.
Early definitive detection and classification of cancerous growths is often crucial to successful treatment of this disease. Currently, several biopsy techniques are used as diagnostic methods after cancerous lesions are identified. In the case of breast cancer, lesions are typically identified with mammography or self breast exam. The most reliable method of diagnosis is examination of macroscopic-sized lesions. Macroanalysis is performed in conjunction with microscopic evaluation of paraffin-embedded biopsied tissue which is thin-sectioned to reveal microscale morphology.
The detection and diagnosis of cancer is typically accomplished through the use of optical microscopy. A tissue biopsy is obtained from a patient and that tissue is sectioned and stained. The prepared tissue is then analyzed by a trained pathologist who can differentiate between normal, malignant and benign tissue based on tissue morphology. Because of the tissue preparation required, this process is relatively slow. Moreover, the differentiation made by the pathologist is based on subtle morphological differences between normal, malignant and benign tissue based on tissue morphology. For this reason, there is a need for an imaging device that can rapidly and quantitively diagnose malignant and benign tissue.
Alternatives to traditional surgical biopsy include fine needle aspiration cytology and needle biopsy. These non-surgical techniques are becoming more prevalent as breast cancer diagnostic techniques because they are less invasive than biopsy techniques that harvest relatively large tissue masses. Fine needle aspiration cytology has the advantage of being a rapid, minimally invasive, non-surgical technique that retrieves isolated cells that are often adequate for evaluation of disease state. However, in fine needle biopsies intact breast tissue morphology is disrupted often leaving only cellular structure for analysis which is often less revealing of disease state. In contrast, needle biopsies use a much larger gauge needle which retrieve intact tissue samples that are better suited to morphology analysis. However, needle biopsies necessitate an outpatient surgical procedure and the resulting needle core sample must be embedded or frozen prior to analysis.
A variety of “optical biopsy” techniques have potential as non-invasive, highly sensitive approaches that will augment, or even be alternatives to current diagnostic methods for early detection of breast cancer. “Optical biopsies” employ optical spectroscopy to non-invasively probe suspect tissue regions in situ, without extensive sample preparation. Information is provided by the resultant spectroscopically unique signatures that may allow differentiation of normal and abnormal tissues. Despite years of research and development, two techniques that have not realized their potential are:                (1) fluorescence optical biopsies, which fails due to the nonspecific nature of tissue autofluorescence; and        (2) near-infrared optical diagnostics, in particular non-invasive glucose sensing, which fails due to interference from tissue major components, including predominantly water.        
In contrast to other techniques, Raman spectroscopy holds promise as an optical biopsy technique that is anticipated to be broadly applicable for characterization of a variety of cancerous disease states. A number of researchers have shown that Raman spectroscopy of masses of cells has utility in differentiating normal vs. malignant tissue and differentiating normal vs. benign tissue. In general, the Raman spectra of malignant and benign tissues show an increase in protein content and a decrease in lipid content versus normal breast tissue, demonstrating that cancer disease states impact the chemistry of the tissue.
However, Raman spectroscopy has not been able to differentiate benign vs. malignant tissues due to the spectral similarities of these tissue types. In addition, Raman spectroscopy of breast tissue samples requires large numbers of cell populations. If only a small portion of the cells are cancerous, as in the early stages of lesion development, then Raman spectroscopy of a large number of such cells will be insensitive to the disease. It would be advantageous to have a technique capable of the spatial sensitivity needed for discrimination of cancerous from normal cells in early stage breast cancer diagnosis.
Chemical imaging based on optical spectroscopy, in particular Raman spectroscopy, provides the clinician with important information. Chemical imaging simultaneously provides image information on the size, shape and distribution (the image morphology) of molecular chemical species present within the sample. By utilizing molecular-specific imaging, based on chemical imaging, the trained clinician can make a determination on the disease-state of a tissue or cellular sample based on recognizable changes in morphology without the need for sample staining or modification.
Apparatus for Raman Chemical Imaging (RCI) has been described by the inventors in U.S. Pat. No. 6,002,476, and in co-pending U.S. Non-Provisional Application 09/619,371 filed Jul. 19, 2000 which claims benefit of U.S. Provisional Application 60/144,518 filed Jul. 19, 1999. The above identified US patents, patent applications, and publications are hereby incorporated by reference.